The endoplasmic reticulum-associated protein degradation (ERAD) pathway serves as quality control for cellular glycoprotein folding in the ER by disposing of misfolded glycoproteins through the ubiquitin/proteasome system. HIV-1 Env is heavily N-glycosylated, which intrinsically accelerates its folding and stability, and is required or its functions. N-glycosylation involves a number of enzymes and chaperones in the ER. Noticeably, the glycoprotein folding in the ER is error-prone, resulting in production of misfolded proteins that are toxic to cells, so cells have evolved the ERAD pathway to specifically target misfolded proteins for degradation. GH47 enzymes are class I mannosidases that include ERManI, EDEM1, EDEM2, EDEM3, Golgi ManIA, Golgi ManIB, and Golgi ManIC. They cleave 1,2-lnked mannose residues from N-linked high- mannose glycan precursors during N-glycosylation, and importantly, they also play an indispensable role in ERAD. In general, ERAD is divided into three steps: substrate recognition, cytoplasmic retrotranslocation, and proteolysis GH47 enzymes are engaged in the recognition step and initiate the degradation. It has been suggested that EDEM1 extracts misfolded proteins from the calnexin/calreticulin cycle, and misfolded proteins are targeted to the ER-derived quality control compartment (ERQC) where ERManI is enriched. ERManI and possibly the EDEM proteins then catalyze extensive demannosylation, which constitutes a signal of protein misfolding that in turn activates the ERAD pathway, resulting in misfolded proteins being degraded. Here, we will study the mechanism of how HIV-1 Env is degraded by the ERAD pathway, and we propose the following two specific aims to understand this mechanism: 1) To elucidate the role of ERManI and EDEM proteins in HIV-1 Env degradation. We will study the ERManI activity in ERAD-mediated Env degradation after ectopic expression and identify its critical molecular determinants for its activity. In addition, we will test how EDEM proteins contribute to the ERManI activity using the advantageous CRISPR/Cas9 knockout (KO) technology. 2) To identify the critical ubiquitin E3 ligase for HIV-1 degradation. We will screen the known E3 ligase library in the ERAD pathway using small interfering RNA (siRNA) knockdown to identify the E3 ligase. The identity of the E3 ligase will be further confirmed by CRISPR/Cas9 KO followed by biochemical analyses. These studies will define novel endogenous and potential therapeutically applicable antiretroviral targets, which specifically inhibits Env expression and blocks HIV-1 replication.